Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in New England Seals, United States

We report the spillover of highly pathogenic avian influenza A(H5N1) into marine mammals in the northeastern United States, coincident with H5N1 in sympatric wild birds. Our data indicate monitoring both wild coastal birds and marine mammals will be critical to determine pandemic potential of influenza A viruses.

1 These first authors contributed equally to this article.
We report the spillover of highly pathogenic avian influenza A(H5N1) into marine mammals in the northeastern United States, coincident with H5N1 in sympatric wild birds. Our data indicate monitoring both wild coastal birds and marine mammals will be critical to determine pandemic potential of influenza A viruses. Complete genomes of HPAI H5N1 viruses (GISAID database, https://www.gisaid.org) were compared by using IQ-TREE (https://www.iqtree. org) with the Ultrafast bootstrap (n = 10,000) option and A/chicken/NL/FAV-0033/2021 as a reference. Bootstrap support values >80 are shown at nodes. Red text indicates seal-derived sequences, black text avian-derived sequences from New England and Newfoundland, and blue text indicates avian-derived sequences from Europe . Branches are shaded on the basis of lineage groups: primary lineage from North America, pink; New England-specific lineage A, 1st wave blue, 2nd wave light blue; and New England-specific lineage B, green. All newly reported specimens were collected in the New England region during February-July 2022. Scale bar indicates nucleotide substitutions per site.
Wild birds in New England experienced 2 waves of influenza infections during 2022. The first wave peaked in March and was largely represented by raptor deaths (39.1% influenza-positive birds). A second wave began in June; gull (38% influenza-positive) and eider (26.8% influenza-positive) deaths were most frequently reported during the second wave. Mortality events affected seabird breeding colonies throughout the coastal region during the second wave; 8 islands had >1 bird test positive for H5 (Appendix 1 Table).
During January 20-July 31, 2022, opportunistic nasal, oral, conjunctival or rectal swab samples were collected from 132 stranded seals along the North Atlantic coast from Maine to Virginia (Appendix 2). HPAI virus was not detected in any of the 82 seals that were sampled through May 31, 2022. Concurrent with the second wave of avian infections, increased seal strandings in Maine led to a National Oceanic and Atmospheric Administration declaration of a UME beginning on June 1, 2022, that included 164 harbor and 11 gray seals in Maine during June and July (12). Swab samples were collected from 41 of those animals; 17/35 harbor and 2/6 gray seals were HPAI-positive and were within coastal regions of known and suspected HPAI outbreaks among terns, eiders, cormorants, and gulls (Appendix Figure 1, panel B). Respiratory symptoms were observed with a subset of neurologic cases, although most stranded seals were deceased. The respiratory tract was the most consistent source of reverse transcription PCR-positive samples from affected seals (15/19 nasal, 16/19 oral, 6/19 conjunctiva, and 4/19 rectal samples).
We sequenced influenza A viruses from swab samples, resulting in 71 avian-and 13 seal-derived virus genomes from New England. We performed phylogenetic analysis of sequences from New England and the most closely related available virus sequences by using IQ-TREE (https://www.iqtree. org) (Figure 1; Appendix 1). We classified all but 1 virus as nonreassortant Eurasia 2.3.4.4b viruses and included those in further analyses (Appendix 1 Figures 2, 3). Sequences fell into 4 distinct clusters; 2 lineages were unique to New England. We found single-nucleotide polymorphisms (SNPs) (Appendix 1 Figures 4-7) and amino acid mutations ( Figure   2) by using vSNP (https://github.com/USDA-VS/ vSNP). Most sequences fell within a dominant New England-specific cluster that spanned the first and second waves (lineage A in this study). All secondwave viruses from lineage A exhibited the acquisition of new, shared mutations. That cluster spanned diverse species, including gulls, geese, eiders, raptors, and seals. A small number of raptor-derived sequences clustered with the primary lineage prevalent in North America at the time of sampling. All but 1 sample from the second wave of avian infections fell into either lineage A or a smaller, unique cluster primarily associated with terns (lineage B in this study).
We inferred that >2 spillover events occurred in the seal population during the second wave of avian infections. Of the sequences derived from seals, 11/13 clustered with second wave lineage A ( Figure 1). We found 4 aa changes in specific proteins in both birds and seals that were distinct from the first wave of HPAI (polymerase acidic protein, A70V; polymerase acidic X protein, A62V; hemagglutinin protein, P152S; and nonstructural 1 protein, R67Q) ( Figure 2). Within second wave lineage A, we found 37 aa changes in >1 seal sequence that were infrequent or absent from bird sequences. Most changes were unique; each occurred in only 1 animal. The polymerase basic 2 protein amino acid substitutions, E627K (in seal no. Pv/MME-22-131) and D701N (in seal no. Pv/MME-22-122), previously associated with mammalian adaptation were each present in 1 seal in second wave lineage A. An additional 2/13 seal-derived sequences clustered with lineage B; 10 aa mutations occurred in both bird and seal sequences ( Figure 2). Another 10 aa changes occurred in at least 1 seal sequence that were infrequent or absent in the bird sequences. In contrast to lineage A, most amino acid changes were shared between the 2 seals in lineage B and were derived from animals stranded within the same town and sampled 1 day apart. The polymerase basic 2 protein substitution, D701N, was present in 1 seal from lineage B (Figure 2, seal no. PV12, .
In contrast to outbreaks in agricultural settings, outbreaks of HPAI in wild populations can rarely be managed well through biosecurity measures or depopulation, which is particularly true for large, mobile marine species such as seals. Avian and mammalian colonial wildlife might be particularly affected by influenza A viruses, which could enable ongoing circulation between and within species, providing opportunities for reassortments of novel strains and study of mammalian virus adaptation. Migratory animals might further disseminate the viruses over broad geographic regions. Therefore, the interface of wild coastal birds and marine mammals is critical for monitoring the pandemic potential of influenza A viruses.